Telomeres are DNA protein complexes that contain repeated sequence DNA and specialized telomere-binding proteins. As telomeres are involved in an amazingly diverse array of biological processes (e.g. tumorigenesis, chromosome segregation, senescence and cell cycle checkpoints), formation of the telomeric DNA-protein complex is essential for proper cell growth and development. The goal of this proposal is to understand how the DNA component of a telomere is replicated. This is a key question because the telomeric complex cannot assemble unless the correct terminal DNA structure is generated. Surprisingly little is known about the structure of the DNA at the end of a telomere, or about how it is replicated. However, it is apparent that replication involves many steps in addition to leading and lagging strand synthesis and addition of G-strand repeats by telomerase. We will characterize the steps needed to replicate the telomeres of two ciliates, Euplotes and Tetrahymena. We have chosen to work with ciliates because their large number of telomeres make it possible to perform experiments that are not feasible in yeast or mammalian cells. The structural differences between Euplotes and Tetrahymena telomeres will enable us to view the range of mechanisms involved in telomere replication. The first three specific aims build on our previous work with Euplotes. Aim 1 asks whether the very precise structure of Euplotes telomeres results from the use of telomeric replication origins. Aim 2 explores the function of the novel replication Telomere Protein (rTP) identified by my lab. We will ask if rTP is involved in replication initiation or telomere repeat addition. Aim 3 ascertains whether rTP has a helicase or DNA strand separating activity. Aim 3 also analyzes how dimerization affects DNA binding. The final two specific aims focus on the replication of the longer more heterogeneous telomeres from Tetrahymena. Since relatively little is known about their terminal DNA structure, aim 4 establishes the structures (long versus short G-strand overhangs) present at different stages in the cell cycle. Aim 5 determines how the G-strand overhangs and previously described C-strand gaps are generated. This analysis will elucidate the steps necessary to generate a functional telomere.